Abstract
Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg(320) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(271), yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg(271). We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg(271), accumulates on the surface because of a approximately 20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.
Highlights
Ability of prothrombin to bind to these membranes through the fragment 1 (F1)2 domain present at its N terminus [1, 3, 4]
Based on the possible concentrations of the various species expected during the activation of prothrombin, these findings suggest that noncovalent interactions with the fragment 2 (F2) domain either on its own or within fragment 1.2 (F12) probably play a minor role in retaining thrombin on the membrane surface or in modulating the function of thrombin in solution
Limiting scattering intensities in line with those expected for vesicle-bound F1 or F12 suggest that the bulk of thrombin produced readily dissociates from the membrane surface even when F12 is produced as a stable product
Summary
F1, fragment 1; DAPA, dansyl-L-arginine-N-(3ethyl-1,5-pentanediyl)amide; FPRck, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; F12, fragment 1.2; F2, fragment 2; II, prothrombin; IIA195, recombinant II containing Ala in place of the catalytic Ser; IIPD, II isolated from plasma; IIQ155/Q284, recombinant II containing Gln substitutions at Arg155 and Arg284; IIQ320, recombinant II containing Gln in place of Arg320; IIQQ, recombinant II containing Gln in place of Arg320 and Arg271; IITM, a triple mutant of II containing Gln in place of Arg at positions 155, 284, and 271; IIWT, recombinant wild type II; IIai, thrombin inactivated with FPRck; mIIa, meizothrombin; mIIai, mIIa inactivated with FPRck; P2, prethrombin 2; IIaA195, thrombin produced from IIA195; P2A195, P2 produced from IIA195; PC, L-␣-phosphatidylcholine; PS, L-␣-phosphatidylserine; PCPS, small unilamellar vesicles containing 75% (w/w) PC and 25% (w/w) PS; PCPSLUV, large unilamellar PCPS vesicles; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid. Based on the possible concentrations of the various species expected during the activation of prothrombin, these findings suggest that noncovalent interactions with the F2 domain either on its own or within F12 probably play a minor role in retaining thrombin on the membrane surface or in modulating the function of thrombin in solution. Thrombin released by cleavage at Arg155 would be expected to be essentially saturated with F2 bound to the anion binding exosite II region of the proteinase This interaction is known to significantly impact active site function, inhibition of thrombin by antithrombin III and possibly platelet aggregation [6, 15,16,17]. The proposed tight interactions with F2 have unexpected and important implications for the numerous functions
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